Genetic Testing
How Are Chromosome Differences Diagnosed?
A doctor may suspect a chromosome difference or disorder in several different circumstances:
- When a couple is having difficulty conceiving or have experienced multiple miscarriages;
- During pregnancy, when congenital disabilities are detected in the fetus by ultrasound, or right after the birth of a baby with congenital disabilities; or
- During infancy or early childhood, when a child is evaluated for developmental delays or other medical problems.
To test for a chromosome difference, a variety of laboratory techniques are used. A blood test is typically required, but since chromosomes are in nearly all cells in our body, sometimes other samples are used. For example, some labs can use cells from inside the mouth (called a buccal sample) to look at chromosomes. For prenatal testing (that is, testing of a fetus while still in the mother’s womb), cells from the amniotic fluid or placenta are used.
Until 2006 or so, the most common way to look at chromosomes in the lab was by doing a karyotype. Karyotyping takes place in a specialized lab called a cytogenetics laboratory (cyto means cell). This type of lab is typically located in major medical centres associated with a university, but larger community hospitals may have one as well. The lab receives a sample from the patient, and the cells from this sample are then cultured (grown in a dish under sterile conditions, with a mixture of nutrients and hormones that stimulates the cells to grow and divide), which takes a few days. Once enough cells are present, they are treated with a chemical that stops their division at a particular point in the cell called metaphase. At this point, chromosomes are easiest to see under the microscope because they are stretched out and long instead of tightly wound and short. The cells are then burst open on a microscope slide, which releases all the chromosomes onto the slide, where they are “fixed” or stuck into place. A dye called Giemsa stain is then applied so we can see the chromosomes more easily. This process gives rise to the banding pattern we see in the karyotype. Because of the Giemsa stain used, it is called G-banding.
The Process
This entire process, from blood sample to G-banding, takes at least a week. But the hardest part is yet to come. The cytogenetic technologist now has to analyze the chromosomes under the microscope. This technologist is highly trained—he or she spends countless hours looking at the chromosomes learning how to identify them by their size and banding pattern. For each sample, the technologist must count the chromosomes, to make sure all 46 are present. Then, she looks at each chromosome, band by band, to ensure that there are no pieces extra (duplicated) or missing (deleted), or any pieces that have swapped places (translocated). Then, she must do this whole process over again, but for seven to ten additional cells in the same sample! So, as you can now imagine, the process of chromosome analysis by karyotyping is a long, laborious process that is prone to technical and human error.
There are other ways to diagnose chromosome differences. One way to look for specific chromosome disorders, such as microdeletion and microduplication syndromes, is called fluorescence in situ hybridization (FISH for short). For this test, the doctor must suspect a particular diagnosis, for example, Williams syndrome, which is caused by a deletion on chromosome 7. This deletion is small enough that you cannot see it by looking at the chromosomes under a microscope. To detect this tiny deletion, the lab uses a FISH probe, which is a piece of DNA that exactly matches the region of the chromosome we are interested in, to “stick” (hybridize) to the chromosomes from the patient. The probe is labelled with a fluorescent dye, so the technologist can see it using a special microscope. A sample taken from a person with normal chromosomes will have two bright spots in the region where the probe sticks—remember, there are two copies of chromosome 7 in nearly every cell in the body. If the patient is missing that particular piece of chromosome 7, the technologist will see only one bright spot.
Chromosomal Microarray (CMA)
The most advanced laboratory technique for the diagnosis of chromosome differences used today is called chromosomal microarray (CMA for short). There are many different methods for doing this same type of test—you may see different names such as microarray, SNP array, and oligo aCGH. This technique allows the laboratory scientist to look at all the chromosomes at once, like a karyotype, but it can detect much smaller imbalances.
To do a chromosomal microarray, DNA is extracted from the patient’s blood sample and purified so that all the other parts of the chromosomes, such as the proteins that hold them together, are washed away. The DNA from the patient is “chopped up,” and what happens next depends on the precise technique used in the lab. But the result is the same—the patient’s DNA is then compared to a control sample (usually, this is a mixture of DNA from a large number of people without major chromosome differences). Here is the idea: if a patient’s sample has no chromosome differences (that is—no pieces missing or extra), then when mixed with the control sample, all the pieces will match exactly. But, if the patient has a deletion or duplication of part of a chromosome, there will be an imbalance in the DNA sample, detected by a very sophisticated computer program. The computer can pinpoint exactly where the imbalance is (i.e., which chromosome), how big it is, and whether it is a deletion or duplication.
Chromosomal microarray has replaced the karyotype as the best test for diagnosing chromosome differences. It is rare for doctors to use karyotype or FISH testing these days. There are some scenarios in which a karyotype is a better test (such as for confirming that a person has Down syndrome, or when looking for a balanced chromosome difference, like a translocation).
CMA is so sensitive for detecting small chromosome imbalances that scientists were initially surprised to find out that all of us have pieces of chromosomes missing or extra when compared to each other. With the increased use of this technology to test millions of people, it has become clear that most of these differences are tiny and do not disrupt any important genes. These benign chromosome differences are called copy number variations, or CNVs for short. When the hospital laboratory reports the results of a CMA, you will not find out about these tiny CNVs, because they are considered a normal part of human variation. The laboratory will only report rare or significant changes. Sometimes these more considerable changes are definitely the cause of developmental disability or other health problems. We know this because, if we compare people with the same finding, they all have similar problems. Sometimes, they even look alike. It is also very common to find CNVs that are large and rare, but when you compare people with the same finding, they are all very different. These CNVs are variants of uncertain clinical significance because we can not know for sure if they are causing the problem. If one of these uncertain changes is found in your child, your doctor may ask for both parents to give a blood sample to see if the change is inherited from mom or dad. If so, the change is less likely to be significant. If the change is spontaneous—not inherited from either parent—this is called a de novo change, and it is more likely to be significant. However, until enough people with the same findings are studied, we cannot know for sure.
Written by Dr. Melissa Carter.
Excerpt from Raising the Goddess of Spring: A guide for parents raising children with rare chromosome disorders Available on Amazon.
Learn More: Genetic Testing Current Approaches – National Library of Medicine
